PHYSIOLOGY,BIOCHEMISTRY, AND TOXICOLOGY Digestive Enzymes and Stylet Morphology of Deraeocoris nebulosus (: ), a Predacious Plant Bug

1 2 DAVID W. BOYD, JR., ALLEN CARSON COHEN, AND DAVID R. ALVERSON

Department of Entomology, Clemson University, Clemson, SC 29634

Ann. Entomol. Soc. Am. 95(3): 395Ð401 (2002) ABSTRACT Mixed-feeding habits, such as zoophytophagy, make the ecological roles of many of , especially hemipterans, difÞcult to assess. To understand the feeding adaptations of the predaciousplant bug Deraeocoris nebulosus (Uhler), the digestive enzymes from the salivary glands and anterior midgut were analyzed, and the mouthpart stylets were investigated with scanning electron microscopy. Evidence of trypsin-like enzyme, ␣-glucosidase, and pectinase were found in the salivary glands. Low levels of trypsin-like, chymotrypsin-like, elastase-like, and pectinase activity, with high levelsof ␣-amylase and ␣-glucosidase activity, were found in the anterior midgut. The insectÕs right maxillary stylet has two rows of at least six recurved barbs on the inner surface pointing away from the head. Thisplant bug isequipped mainly for zoophagy but hasenzymesthat would allow some degree of phytophagy.

KEY WORDS Deraeocoris nebulosus, digestive enzymes, Miridae, predator, stylet morphology, omnivory

FEEDING HABITS OF the range from strict A theoretical and practical grasp of the role that phytophagy to strict zoophagy (Schaefer and Panizzi heteropterans play in natural and agricultural systems 2000). Several familiescontain omnivoreswhose requiresa thorough understandingof their feeding mixed-feeding habitshave been termed zoophytopha- habits. Such knowledge, however, is difÞcult to obtain gousor phytozoophagous,depending on the relative by direct means, largely because of the cryptic nature degree of versus plant consumption (Alomar of the feeding processandthe amorphousnature of and Wiedenmann 1996). The origin of feeding habits the ingested food. Heteropteran workers lament the among the Heteroptera remainscontroversial(Sweet lack of a thorough understanding of heteropteran 1979, Cobben 1979, Cohen 1990, Schaefer 1997, feeding habits, especially for the Miridae (Wheeler Wheeler 2001). The diverse trophic habits of plant 2001). bugsmake the Miridae ideal for studiesoffeeding A consumerÕs ability to use plant or animal materials strategies, including digestive enzyme composition for food is indicated by the presence of speciÞc di- and mouthpart morphology. gestive enzymes and by mouthpart morphology (Bap- Mirids,aswell asall other heteropterans,employ tist 1941; Adams and McAllan 1956; Strong and Kruit- macerate (or lacerate) and ßush feeding (Miles 1972, wagen 1968; Miles1972; Cohen 1990, 1995, 1996, 1998a, Hori 2000, Wheeler 2001) that incorporatespiercing/ 1998b, 2000; Agustõ´ and Cohen 2000; Hori 2000; Zeng sucking mouthparts and watery saliva from the sali- and Cohen 2000a, 2000b). Digestive enzymes speciÞc vary gland complex. Miridsfeed in a manner that is for zoophagy include proteases (e.g., trypsin, chymo- typical of heteropterans, piercing and cutting tissues trypsin, cathepsin), hyaluronidase, and phospholipase with their stylets while injecting digestive enzymes (Cohen 1998b, 2000). SpeciÞc digestive enzymes for through the salivary canal to liquefy food into a nu- phytophagy include amylase and pectinase (Cohen trient-rich slurry. The food slurry is ingested through 1996). the food canal and passed into the alimentary canal Morphological comparisons of both mandibular and where it is further digested and absorbed (Cohen maxillary styletsrevealdifferencesbetween heterop- 2000). teran phytophagesand zoophages.Cobben (1978) showed that the right maxillary stylets of predacious Thisarticle reportsthe resultsofresearchonly. Mention of a heteropterans(e.g., Nabidae, Anthocoridae) are more proprietary product does not constitue an endorsement or a recom- deeply serrated than those of phytophagous heterop- mendation by the USDA for itsuse. terans(e.g., Tingidae). Cohen (1996) showedthat the 1 Current address: Small Fruit Research Station, USDA-ARS, P.O. Box 287, Poplarville, MS 39470 (e-mail: [email protected]). mandibular styletsofphytophagousand predacious 2 Biological Control and Mass Rearing Research Unit, USDA-ARS, pentatomidsvaried in relation to the direction of the P.O. Box 5367, Mississippi State, MS 39762. barbs; those of phytophagous species point away from

0013-8746/02/0395Ð0401$02.00/0 ᭧ 2002 Entomological Society of America 396 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 95, no. 3

the head, whereasthe barbsof predaciousspecies natant wasplaced in a 1.5-ml centrifuge tube and kept point toward the head. The barbson the mandibular at 4ЊC until use (within 48 h). The anterior midguts of styletsofpredaciousheteropteran families(e.g., the same 25 or 10 insects were treated as were the ) are more numerousthan the barbson the salivary glands. Protein concentrations of all enzyme mandibular styletsofphytophagousheteropteran samples were determined by bicinchoninic acid pro- families (e.g., Lygaeidae, sensu lato) (Cohen 1990). tein assay (Pierce, Rockford, IL), using bovine serum Deraeocoris nebulosus (Uhler) isa predator of plant- albumin as the standard. Three samples from 25 or 10 feeding including aphids, mites, scale in- insects were used for each tissue. sects, and whiteßies (Gillette 1908, Smith 1923, ␣-Amylase Assay. Amylase activity in the salivary Wheeler et al. 1975, Jonesand Snodgrass1998).Phy- glandsand anterior midgut wasdetermined with a tophagy isunknown in D. nebulosus, but other species diagnostic kit (No. 577Ð3, Sigma, St. Louis, MO) fol- in thispredatory genusfeed facultatively on plants lowing modiÞcationsof Zeng and Cohen (2000a, (McMullen and Jong 1967, RazaÞmahatratra 1981, 2000b, 2000c). The substrate was 4,6 ethylidene (G7)- Wheeler 2001). One of us(D.W.B.) hasobserved D. p-nitrophenyl (G1)-␣, D-maltoheptaside. Enzyme ex- nebulosus with its stylets inserted into sweet potato tracts(10 ␮l) were added to wellsin an ELISA plate. leavesand cabbage, but whether thisbehavior in- The substrate was equilibrated to 37ЊC for 10 min, and volvesthe uptake of nutrientsor justwater isnot 200 ␮l wasadded to each well. The plate wasshaken known. for Þve sand incubated at 37 ЊC for 30 min. Absorbance The objectivesof thisstudywere to determine the wasread at 405 nm in a plate reader (SPECTRA MAX presence of certain digestive enzymes in the salivary Plus, Molecular Devices Corporation, Sunnyvale, gland complex and anterior midgut of D. nebulosus and CA). Absorbance is directly related to amylase activ- to analyze its stylet morphology. We also evaluated ity. Authentic ␣-amylase from malt (Sigma thispredaciousmiridÕsability to obtain nutrientsfrom A-2771) wasusedasa positivecontrol (1 mg/ml, 1 animalsand plants. U/mg solid), and buffer and substrate only were used asa negative control. The relative amylaseactivity was calculated asabsorbanceunitsper milligram of pro- Materials and Methods tein. The assay was performed three times for each Insects. A colony was established with D. nebulosus sample. collected from oaks(e.g., Quercus alba L., Q. stellata ␣-Glucosidase Assay. ␣-Glucosidase activity was Wang, and Q. falcata Michaux) in Greenville County tested from both salivary gland and anterior midgut and PickensCounty, SC, during the summerof 1999 extracts, following Agustõ´ and Cohen (2000), by add- and wasmaintained at 25 Ϯ 2ЊC, 50 Ϯ 10% RH, and a ing 100 ␮l of extract to 100 ␮l of 10-mM solution of photoperiod of 14:10 (L:D) h. Deraeocoris nebulosus p-nitrophenyl ␣-d-glucopyranoside (Sigma, N-1377) wasreared on eggsof Ephestia kuehniella Zeller (Lep- in an ELISA plate and incubating at 37ЊC for 1 h. The ␮ idoptera: Pyralidae) (BeneÞcial Insectary, Redding reaction wasstoppedby adding 100 l of 15% Na2CO3. CA, USA) at the Cherry Farm Insectaries, Clemson Invertase from bakers yeast (Sigma I-4504) was used University, Clemson, SC, USA. Voucher specimens as a positive control (1 mg/ml, 400 U/mg solid), and were placed in the Clemson University buffer and substrate only were used as a negative Collection. control. Absorbance was read at 405 nm in a plate Sample Preparation. Enzyme samples were pre- reader, and the relative ␣-glucosidase activity was pared by the method of Cohen (1993) with modiÞ- calculated asabsorbanceunitsper milligram of pro- cation. Only adult female insects were used in these tein. The assay was replicated three times. tests. The mirids were starved for 24 h before dissec- General Protease Assay. General protease activity tions to standardize the insects and to allow an accu- was determined using the EnzChek ßuorescence pro- mulation of digestive enzymes. The insects were tease assay kit (E-6638, Molecular Probes, Eugene, placed at Ϫ20ЊC for 4 min and then dissected in ice- OR) following modiÞcationsof Zeng and Cohen cold phosphate buffer saline (pH 7.4) under a dis- (2000a). Extract samples were diluted by adding 10 ␮l secting microscope. The salivary gland complex, in- of extract to 90 ␮l of the reaction buffer in an ELISA cluding all lobes, accessory glands, and tubules, was plate well, and then 100 ␮l of substrate solution buffer exposed by holding the abdomen with Þne forceps and (casein derivatives heavily labeled with the pH-insen- pulling the head and away from the abdo- sitive BODIPY FL-dye) was added. The assay plate men with another pair of Þne forceps. The anterior wasincubated and protected from light fo r1hatroom midgut wasexposedby holding the body with forceps temperature (Ϸ25ЊC). Fluorescence, which is propor- and pulling the ovipositor and last three or four seg- tional to protease activity, was measured in a ßuores- mentsof the abdomen away from the restof the cent plate reader (SPECTRA MAX Plus, Molecular abdomen with another pair of forceps. DevicesCorporation, Sunnyvale, CA), equipped with The salivary glands of 25 insects were removed and standard ßuorescein Þlters, at excitation/emission, placed in 1 ml of phosphate buffer for all assays except 485/538 nm. Authentic trypsin from bovine pancreas pectinase and hyaluronidase assays, where 10 insects (Sigma T-8003) and buffer and substrate only were were used, and the tissues were placed in 400 ␮lof used as positive (1 ml/mg, 10,000 BAEE units/mg buffer. The tissues were homogenized and then cen- protein) and negative controls, respectively. Relative trifuged at 15,294 ϫ g for 10 min at 4ЊC. The super- proteaseactivity wascalculated asßuorescenceunits May 2002 BOYD ET AL.: DIGESTIVE ENZYMES OF D. nebulosus 397 per milligram of protein. The assay was performed three timesfor each sample. Specific Protease Assays. Trypsin-like, elastase-like, and chymotrypsin-like enzymes were analyzed from salivary glands and anterior midguts, following the methodsof Agustõ ´ and Cohen (2000), using 10 ␮Mof speciÞc nitroanilides for trypsin, elastase, and chymo- trypsin, respectively. The substrates for trypsin and elastase, N␣-benzoyl-l-arginine-p-nitroanilide (Sigma B-3133) and succinyl-alanyl-alanyl-alanyl-p-nitroani- lide (Sigma S-4760), respectively, were dissolved in dimethylsulfoxide (DMSO) and added to phosphate buffer to make a 10-␮M solution. The substrate for chymotrypsin, benzyol-l-tryosine-p-nitroanilide (Sig- ma B-6760), was dissolved in dimethylformamide Fig. 1. Anterolateral view of the stylet bundle of Deraeo- (DMF) and added to phosphate buffer to make a 10 coris nebulosus. The two mandibular stylets are outside the ␮M solution. In ELISA plate wells 50 ␮l of each extract two maxillary stylets. Scale bar ϭ 50 ␮m. wasadded to 200 ␮l each of the three substrates. Trypsin from bovine pancreas (Sigma T-8003), elas- tase from porcine pancreas (Sigma E-0258), and coated with a sputtering device and viewed in sec- ␣-chymotrypsin from bovine pancrease (Sigma ondary emission mode in a Hitachi 3500 scanning C-7762) were used as the positive controls (1 mg/ml; electron microscope at 10 kV. 10,000 BAEE units, 3Ð6 U, and 40Ð60 U/mg protein, respectively) and buffer and substrate only was used Results asa negative control. After a 20-h incubation at 37 ЊC, absorbance was read at 405 nm in a plate reader and Digestive Enzymes. Amylase activity was detected the relative protease activity for each substrate was in the anterior midgut (0.85 Ϯ 0.05 SE, n ϭ 9) but not calculated asabsorbanceunitsper milligram of pro- in the salivary gland complex, and glucosidase activity tein. The assays were performed three times for each wasfound in the anterior midgut (0.91 Ϯ 0.04 SE, n ϭ sample. 3) and salivary gland complex (0.11 Ϯ 0.13 SE, n ϭ 3). Pectinase Assay. Pectinase activity was tested both General protease activity was observed from the an- from salivary gland and anterior midgut extracts fol- terior midgut (99.89 Ϯ 12.29 SE, n ϭ 9) and salivary lowing Agustõ´ and Cohen (2000) with slight modiÞ- gland complex (4,491.44 Ϯ 304.13 SE, n ϭ 9). cation. Extract consisting of 100 ␮l wasadded to 4 ml Trypsin-like activity was detected in the salivary of 0.5% pectin from citrusfruits(Sigma P-9135) in gland complex (1.90 Ϯ 0.44 SE, n ϭ 3) and anterior phosphate buffer saline (pH 7.4) and incubated at midgut (0.04 Ϯ 0.01 SE, n ϭ 3). Elastase-like and 37ЊC for 20 h. The pectin-extract solution was passed chymotrypsin-like activities were found from the an- through a #150 Cannon-Fenske routine type viscom- terior midgut (0.07 Ϯ 0.04 SE, n ϭ 3 and 0.03 Ϯ 0.02 eter (Cannon Instrument, State College, PA) at 25ЊC. SE, n ϭ 3, respectively) but not from the salivary gland Heat denatured extract wasusedasa negative control. complex. Pectinase activity was measured as the reciprocal of Pectinase activity was found in the salivary gland viscosity (1/centistokes) per milligram of protein, be- complex (0.74 Ϯ 0.02 SE, n ϭ 3) and in the anterior cause pectinase activity is indicated by viscosity re- midgut (0.18 Ϯ 0.04 SE, n ϭ 3). Hyaluronidase was not duction rather than a direct reading of viscosity. detected in either the salivary gland complex or the Hyaluronidase Assay. Hyaluronidase activity was anterior midgut. tested from both salivary gland and anterior midgut Mouthpart Morphology. The stylet bundle of D. extracts similarly to pectinase. A 0.2% solution of hy- nebulosus hastwo maxillary styletsinsidetwo man- aluronic acid from rooster comb (Sigma H-5388) in dibular stylets (Fig. 1). The insectÕs right maxillary phosphate buffer saline (pH 7.4) was used as the stylet has two rows of at least six recurved barbs on the substrate. The reciprocal of viscosity was used to ex- inner surface pointing away from the head and the press enzyme activity. barbsare longer closerto the tip (Fig. 2). Scanning Electron Microscopy Preparation. Six- teen heads, including mouthparts, of D. nebulosus Discussion were placed in 95% ethanol for at least 24 h and then air dried. Dry headswere placed, labium up, on alu- Proteases. The presence of protease activity in the minum stubs with double-sided tape. Stylets were re- salivarygland complex of thispredator wasexpected. moved from the labium with an pin. Stylet Trypsin-like protease was the only protease detected bundleswere kept together or were separatedinto in the salivary gland complex, but the anterior midgut their mandibular and maxillary stylets by placing a had low levels of trypsin-like, elastase-like, and chy- minuten pin at the base of the stylets and moving it motrypsin-like proteases. The presence of trypsin-like toward the apex or by sliding forceps gently over the enzymesdemonstratestheinsectÕsabilityto access stylets from the base to the apex. Specimens were gold structural or other insoluble proteins (Cohen 1993, 398 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 95, no. 3

teinsonce they are insidethe gut, making the proteins absorbable after further hydrolysis by exopeptidases such as aminopeptidase and carboxypeptidase. Amylase. The salivary gland complex has no detect- able amylase activity. The anterior midgut has approx- imately equal levelsof ␣-amylase and ␣-glucosidase activity. Deraeocoris nebulosus might ingest intact starch granules, with starch digestion occurring com- pletely in the midgut. Also, the ingestion of soluble glycogen from prey may be an alternative or additional explanation for the presence of amylase in the gut and its absence in the salivary glands. The distribution of amylase in either or both the salivary glands or guts of heteropteransin variousecological nichesremainsto Fig. 2. Lateral view of the right maxillary stylet of De- be clariÞed. Conversely, plant-feeding mirids usually raeocoris nebulosus. Scale bar ϭ 50 ␮m. have high levels of amylase in the salivary glands (e.g., Agustõ´ and Cohen 2000). These amylase secretions are thought to be ingested by the mirid along with par- 1998a, 2000). Possession of trypsin-like enzymes and tially digested starches to be used in the midgut to the lack of elastase-like and chymotrypsin-like en- continue the breakdown of starch (Hori 1973, Ta- zymesin the salivarygland complex iscommon for kanona and Hori 1974, Wheeler 2001). The lack of heteropteran predators(Cohen 1993, 1998a, 2000; detectable amylase in the salivary glands of D. nebu- Agustõ´ and Cohen 2000). Trypsin-like enzymes are losus indicatesthat plant material isnot a large part of endoproteasesthatattack proteinsat residuesofar- itsdiet; thisfundamental enzyme isfound in the sal- ginine and lysine, but chymotrypsin-like enzymes, also ivary glandsof phytophagousheteropterans(Hori endoproteases, attack proteins at aromatic residues 2000). Deraeocoris pulchellus (Reuter) [as D. punctu- (e.g., tryptophan). Elastase-like protease has been latus (Falle´n); see Kerzhner and Josifov (1999)] has identiÞed only in the salivary glands of one mirid, “very low activity” of amylase in its salivary glands Lygus hesperus Knight (Zeng and Cohen 2001) and in (Hori 1972). Deraeocoris nigritulus, a predator found the salivary glands of the reduviid, renardii Ko- on Pinus virginiana Miller hasno detectable amylase lenati (A.C.C., unpublished data). These authors activity in itssalivaryglands(D.W.B., unpublished found more elastase activity in Þeld-caught insects data). than those reared in the laboratory. Individuals of D. The mainly predaciousgeocorid, Geocoris punctipes nebulosus in thisstudywere reared in the laboratory, (Say), which uses plant material for nutrients, has but another species, D. nigritulus Knight, wasÞeld amylase activity in its salivary glands, indicating its collected and in a similar study also had no detectable ability to digest starches of plants before ingestion elastase activity (D.W.B., unpublished data). (Zeng and Cohen 2000a). Orius insidiosus (Say), a The strictly phytophagous mirid Poecilocapsus lin- predaciousanthocorid, hasamylaseactivity, detected eatus (F.) lacks detectable digestive proteases in its by whole-organism homogenization, demonstrating salivary gland complex (Cohen and Wheeler 1998), its ability to digest starch (Zeng and Cohen 2000b). indicating an inability to use animal protein. However, Cohen (1996) showed that the predators Nabis alter- the phytozoophagousmirids Lygus lineolaris (Palisot natus Parshley (Nabidae) and Sinea confusa Caudell de Beauvois) and L. hesperus have trypsin-like and (Reduviidae) lack amylase activity in their salivary elastase-like enzymes but not chymotrypsin-like en- glands; another reduviid, Zelus renardii, shows amy- zymesin their salivarygland complexes(Agustõ ´ and lase activity in its salivary glands. Although closely Cohen 2000), demonstrating their ability to use animal related species often have similar enzymes, each spe- protein for nutrients. Lygus hesperus producesmore cies should be studied individually for the presence or elastase when given an elastin-spiked artiÞcial diet absence of enzymes. (Zeng and Cohen 2001). The green mirid ␣-Glucosidase. The presence of ␣-glucosidase in the dilutus (Stål) was the Þrst mirid shown to have chy- salivary glands suggests that D. nebulosus can break motrypsin-like activity in the salivary glands down the sugary water from plants before ingestion, (Colebatch et al. 2001). The trypsin-like enzyme se- unlessthisenzymeisa nonsecretablecellular enzyme creted into the food from the salivary glands through of the salivary glands as suggested by some heterop- the salivary canal is thought to be ingested by the teran studies(Hori2000). Twelve other miridswere predator along with food and employed in the gut to found to have low levels of glucosidase in the salivary continue breakdown of proteins(Cohen 1998b). glands(Agustõ ´ and Cohen 2000, Hori 2000). Because Low levels of activity of all three speciÞc proteasesÐ the substrates of ␣-glucosidase are water soluble and trypsin-like, elastase-like, and chymotrypsin-likeÐin commonly found might explain why only low levelsof the anterior midgut indicate either the ability of D. thisenzyme are found in the salivaryglandsof many nebulosus to synthesize proteases in the gut or the heteropterans. One notable exception is the coreids, presence of symbiotic microorganisms in the gut. which have strong glucosidase activity (Taylor and These alkaline endoproteases help break down pro- Miles1994, Hori 2000). May 2002 BOYD ET AL.: DIGESTIVE ENZYMES OF D. nebulosus 399

Pectinase. The presence of pectinase in the salivary representing an intermediate condition (Wheeler glandsand midgutsindicatesan ability to feed on 2001). Boyd (2001) observed maxillary stylets from 20 plantsby attacking cellsheld together with pectin mirids, ranging from strictly phytophagous to strictly complexes. This enzyme, supposedly characteristic of zoophagous, and suggested that the serrations of the the Miridae (Takanona and Hori 1974, Wheeler 2001), right maxillary styletsaredeeper for predatorsthan for might indicate that D. nebulosus hasevolved from a herbivores. The deep serrations in the right maxillary phytophagous species. Hori (2000) suggested that, stylet of D. nebulosus (Fig. 2), which are similar to among the Heteroptera, pectinase is a fundamental those of D. olivaceous (F.) (Cobben 1978), probably enzyme only in the Miridae. Pectinase might play a are used to disrupt prey by ripping and tearing tissues signiÞcant role in egg-laying behavior. Nearly all (Cohen 2000). The barbspoint away from the head known miridslay their eggsin plant material; some which indicate that the cutting action occurswhen the depositeggson plants(Wheeler 2001). Preoviposition stylet is thrust forward, unlike predacious pentatomids behavior includesprobing the plant with the ros- trumÑsometimes for more than 10 min (Knight which have barbson the mandibular styletspointing 1941)Ñbefore placing the ovipositor in the plant ma- toward the head (Cohen 1996) terial (Ferran et al. 1996, Wheeler 2001). Hori and Conclusions. Deraeocoris nebulosus hasan arsenalof Miles(1993) found “strong” pectinase in saliva dis- digestive enzymes suitable for zoophagy. The salivary charged without stimulation from the tip of the stylets enzymes include trypsin-like protease, which is es- of Creontiades dilutus (Stål) (Hori 2000). Pectinase sential in breaking down otherwise insoluble proteins found in mirid zoophages, such as D. nebulosus and D. in the prey. These enzymes and the deep serrations of nigritulus (D.W.B., unpublished data), might be used the maxillary styletsenablethispredator to break to soften the plant material before oviposition. This down tissues of arthropods mechanically and chemi- possible function merits further study. cally (Cohen 2000). The presence of ␣-glucosidase Hyaluronidase. Hyaluronidase is a common com- and pectinase in the salivary glands indicates the abil- ponent of the venoms of scorpions, spiders, and at least ity of D. nebulosus to use plant material for more than one reduviid, Platymeris rhadamantus Gaerst (Ed- just water uptake. wards1961). Asa component of venom, it disrupts The presence of ␣-amylase activity in the midgut extracellular matrix and basement membranes, allow- indicatesthe ability of D. nebulosus to digest starch ing venoms to disburse throughout organs and tissues. from plants. ␣-Glucosidase in the midgut enables the Hyaluronidase is used to disrupt basement membranes bug to break down sugar obtained from starch or of organs and tissues inside the prey, acting as a tissue- soluble glycogen from prey into monomeric units for macerating enzyme (Cohen 1998a, 1998b). The ab- absorption. The ability to use plant material might sence of this enzyme, therefore, raises questions about D. nebulosus how D. nebulosus gainsaccesstonutrientsfrom the enable to survive in the absence of prey tissue matrix. Other types of tissue-macerating en- for short periods of time, which would enhance the zymesmight be employed by thisspecies,including potential of thisbug asa biological control agent chondroitinases or collagenase, or D. nebulosus might (Naranjo and Gibson 1996). use predominantly mechanical means of tissue mac- Deraeocoris nebulosus isequipped mainly for zooph- eration. The presence of a deeply serrated rasp on the agy but hasenzymesthat would allow somedegree of right maxillary stylet of D. nebulosus (Fig. 2, see dis- phytophagy. Thismirid isa voraciouspredator of cussion below) might compensate for the lack of hy- many species of arthropods and might be able to sus- aluronidase. The rasp might mechanically disrupt the tain itself on plant material for short periods of time. organs and tissues, while the enzymes the mirid does Itsfurther evaluation asa biological control agent is have (e.g., trypsin) further macerate the tissue chem- warranted. ically (Cohen 2000). Stylets. The mandibular stylets of D. nebulosus (Fig. 1) are typical of the Miridae whether phytophagousor carnivorous(Cobben 1978, Cohen 1996, Boyd 2001, Acknowledgments Wheeler 2001). Many other heteropteransproduce a salivary ßange that is used as a fulcrum, among other We thank Patrick Crittenden (USDA-ARS) for excellent functions, for stylet movement (Cohen 1998b, 2000). technical assistance, and P. H. Adler, A. G. Wheeler, Jr. (both Because mirids do not produce a salivary ßange, the from Clemson University), Carl Schaefer (University of Con- necticut), J. R. Boyd, and one anonymousreviewer for crit- apical serrations on the mandibular stylets are con- ical reviewsof earlier draftsof the manuscript.We alsothank sidered adaptive in holding onto tissues below the T. Brown and K. Bryson (both from Clemson University) for outer layer of the prey or host plant and in producing allowing usto useequipment in their laboratory for the a fulcrum for movement of the maxillary stylets pectinase and hyaluronidase experiments. Our thanks is ex- (Cobben 1978, Cohen 2000, Wheeler 2001). tended to JoAn Hudson (Clemson University) for instruction Cobben (1978) suggested that the inner surface of in using the scanning electron microscope. This work was the right maxillary styletsofheteropteransranged funded by the W. C. NettlesEndowed Memorial Grant, from moderately serrated in predatory families (e.g., Clemson University (to D.W.B.), and the Ornamental En- Anthocoridae, Nabidae) to smooth in strictly phyto- hancement Program, Clemson University (to D.W.B. and phagousfamilies(e.g., Tingidae), with the Miridae D.R.A.). 400 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 95, no. 3

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